Achieving bone union can be difficult in all aspects of orthopaedics including spine, trauma, and upper extremity surgery. Failure to achieve bone union results in multiple additional surgeries, significant morbidity, and ultimately can lead to loss of extremity function or amputation. To overcome the limitations in current orthopaedic practice, researchers are using tissue engineering techniques. By combining biodegradable scaffolds with bone inducing factors such as bone morphogenetic proteins (BMPs) and demineralized bone matrix, missing bone tissue in segmental defects or non-unions can be healed. BMP-2, though potent in promoting bone regeneration, can lead to heterotopic bone formation which may require additional surgery. Therefore, there is a great need for new candidate molecules that might provide treatment results equivalent to or better than now achieved. Recently we found that thrombopoietin [TPO, the main megakaryocyte (MK) growth factor] could heal critical size mouse femoral defects as well as, if not better than, BMP-2. We hypothesize that TPO accelerates bone healing by two important mechanisms. First, TPO increases MK numbers which in turn enhance OB proliferation, increases bone formation, and accelerates periosteal bridging. Second, TPO more rapidly restores bone contour at the fracture site by binding to c-mpl on osteoclast (OC) progenitors, increasing osteoclastogenesis, and subsequently coupled remodeling. Therefore, in Aim 1, we will establish how TPO impacts bone regeneration in a mouse critical size defect model. In Aim 2, we will dissect the mechanisms by which TPO enhances OB number. In Aim 3, we will determine the mechanisms by which TPO enhances OC number and function. Successful completion of these studies will demonstrate the utility of using TPO to enhance bone healing under difficult conditions such as large segmental defects and nonunion. Further, these studies will begin to provide us with the mechanisms underlying the success of TPO in bone healing. Understanding these mechanisms may provide insight into alternative treatment strategies for bone healing as well as systemic bone loss such as that seen in osteoporosis.